The Immunostimulatory Potential of Mesenchymal Stem Cells for formation of Tertiary Lymphoid Structures in Lupus Nephritis

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Department of Medical Biology - Faculty of Healthy Science

The Immunostimulatory Potential of

Mesenchymal Stem Cells for formation of

Tertiary Lymphoid Structures in Lupus Nephritis

—

Aud-Malin Karlsson Hovd

Master’s thesis in Biomedicine (MBI-3911), May 2017

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Acknowledgements

The work of this master thesis in biomedicine was performed at the RNA and Molecular Pathology (RAMP) research group, department of Medical Biology, Faculty of Health Science at The University of Tromsø – The Artic University of Norway, in the period of August 2016 to May 2017.

First, I would like to thank my main supervisor Kristin A. Fenton for giving me the opportunity to delve deeper into the field of immunology and the molecular pathology of lupus. You saw my potential as a researcher already during my bachelor thesis, and you have motivated me to continue my journey into the world of research. A special thanks goes to my co-supervisor Esmaeil Dorraji, for including me in your PhD project. You have given me insight into the core of what research really is, and you have pushed me to perform my very best.

I also want to give the rest of RAMP research group, and especially Prema, huge and special thanks. You have all made me feel a part of this research group this last year with your positivity and humour!

Thank you to my fellow classmates, for making these two years wonderful. A special thanks goes to my fellow office-mates; Lotte and Siri, your pep talks and coffee breaks have been most welcome.

I would also like to thank my family and my friend Karoline for your endless support, motivation and cheering with a warm and heartfelt “Thank you”. Last but definitely not least, I want to thank my partner Kristine for being there for me with your patience, laugher and love.

Aud-Malin Karlsson Hovd Tromsø, May 2017

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Abstract

Formation of tertiary lymphoid structures (TLS) occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), TLS have been observed in the kidneys of lupus-prone mice and in kidney biopsies of SLE patients with Lupus Nephritis (LN). Here the role of tissue-specific mesenchymal stem cells (MSCs) as lymphoid tissue organizer cells on the activation of CD4+

T cells from three groups of donors; Healthy, SLE patients and LN patients were investigated.

Human MSCs were stimulated with the pro-inflammatory cytokines TNF-α and IL-1β to resemble an inflammatory condition. CD4⁺T cells isolated from PBMC were co-cultured with stimulated and non-stimulated MSCs at 1:1 and 1:100 ratios (MSCs:CD4⁺T cells) or seeded alone as a control. The AlamarBlue® proliferation assay was performed on CD4⁺T cells at day zero and at day 5, 7 and 10 after co-culture. Flow cytometric analyses were conducted on CD4⁺T cells at day zero and day 10 to analyse the Th1, Th2, Th9, Th17, Th22, and Th1/17 subsets before and after co-culturing with MSCs. To detect MSCs within TLS in kidneys of lupus-prone (NZBxNZW) F1 mice confocal imaging was used.

After stimulation a significant increase in the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1β were observed in MSCs. For all groups CD4⁺T cells co-cultured with stimulated MSCs and non-stimulated MSCs at 1:100 ratio proliferated significantly more at day 10 compared to day zero and CD4⁺T cells cultured. CD4⁺T cells co-cultured with stimulated MSCs at 1:100 ratio proliferated significantly more than co-cultured with non- stimulated MSCs at day 10 in healthy and SLE groups, but not in the LN group. No difference in cell proliferation at 1:1 ratio was detected. An increase in Th2 and Th17 subsets were observed in the healthy group at day 10 when co-cultured with stimulated MSCs at 1:100 ratio compared to day zero and CD4⁺T cells alone at day 10. MSC-like cells were detected within the pelvic wall of the kidneys and within the developed TLS.

Our data suggest that tissue-specific MSCs could have pivotal roles in accelerating early inflammatory processes and initiating the formation of TLS in chronic inflammatory condition.

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Abbreviations

ACR American College of the

rheumatology ICAM-1 Intercellular Adhesion

Molecule 1

AIR Annual incidence rate IFN Interferon

ANAs Antinuclear antibodies Ig Immunoglobulin

BCR B cell receptor IL

Interleukin

CCR C-C chemokine receptor LN Lupus nephritis

CD Cluster of differentiation LT Lymphotoxin

CTL Cytotoxic CD8+ T cells MALTs Mucosal associated lymphoid tissues CXCR C-X-C motif chemokine

receptors MHC Major histocompability

complex

DCs Dendritic cells MSCs Mesenchymal stem cells

dsDNA Double stranded

deoxyribonucleic acid SCA-1 Stem cell antigen-1

ECM Extra cellular matrix SLE Systemic lupus

erythematous FcR Fragment-crystallize

receptor SLEDAI SLE disease activity index

FDCs Follicular dendritic cells SLOs Secondary lymphoid organs

FoxP3 Forkhead box P3 TCR

T cell receptor

FRCs Fibroblastic reticular cells TGF Transforming growth factor

GC Germinal centre Th cells CD4+ T helper cells

HEV s High endothelial venules TLR Toll like receptor

HMLE Human mammary

epithelial cells TLSs Tertiary lymphoid

structures

HSCs Hematopoietic Stem cells TNF Tumour necrosis factor

HUV-EC-C Human Umbilical Vein

Endothelial Cells VCAM-1 Vascular cell adhesion protein 1

ICs Immune complexes PBMC Peripheral blood

mononuclear cells

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1 Introduction

1.1 Immune system, tolerance and autoimmunity

The immune system is a creation of the evolution as a defence against the potential danger pathogenic infections generate. The immune system can roughly be divided into two main compartments the innate and the adaptive immune system, where the “communication”

between these parts plays an important role in the development and control of the disease progression. The innate immune system involves most cell types in the body and mediates a general protection against infection, and can for example mediate an activation of the adaptive immune system through antigen presenting cells such as dendritic cells (DCs) and macrophages.

The specificity of the immune system, the power of which a single pathogen are recognized and eliminated, lies within the adaptive immune system. The effector cells of the adaptive immune system are the B and T cells with their respectively B cell receptor (BCR) and T cell receptor (TCR) [1]. With the two processes of selective somatic genome modification; V(D)J recombination of both TCR and BCR in primary lymphoid tissues and somatic hypermutation of the BCR in peripheral lymphoid tissue, receptors with a enormous diversity can be produced [1]. Interesting 20-50% of the V(D)J recombined BCR and TCR can in the theory bind with high affinity and react to a self-antigen, but only 3-8% of the world population is affected by autoimmune diseases [1].

One of the reasons why the percentages of the developing autoimmune diseases in the world population are so low compared to the percentages of autoreactive BCR and TCR, is the tolerance mechanisms involved in the developing lymphocytes and controlling of lymphocytes [1]. There are two main types of tolerance mechanisms acting on the developing lymphocyte: central and peripheral tolerance [2], illustrated in Figure 1.1 and further explained in section 1.4 Lymphoid organs.

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Figure 1.1: Tolerance mechanisms for controlling and removing of autoreactive lymphocytes.

In central tolerance an autoreactive lymphocyte can be removed during their development in either the thymus or in the bone marrow. There are three main mechanisms for central tolerance; BCR change in autoreactive B cells, development of Treg cells or that the autoreactive lymphocyte dies via apoptosis. In peripheral tolerance the autoreactive T or B cell can either be functional inactive in a process known as anergy, die via apoptosis or of autoreactive T cells be controlled by Treg cells.

Modified from ref [2]

Autoimmunity arise when an organisms immune system start to produce an immune reaction against its own cells, tissues and/or organs [3]. The knowledge about the existence of autoimmune diseases has been known for over 100 years with more than 80 human diseases being investigated, but the underlying initiation mechanisms for why autoimmunity exists are still a mystery among scientists [3]. It is clear that there are three main phases of an autoimmune disease; initiation, propagation and resolution (Figure 1.2), which all can be linked to a deficiency in the immune regulatory mechanisms [4].

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Figure 1.2: Common disease progression in autoimmune diseases. Normally when the patients are in the resolution phase, a relapse occurs and acute inflammation with tissue damage are observed [4].

1.2 Systemic Lupus Erythematous

Systemic Lupus Erythematous (SLE) is an autoimmune rheumatic disorder that can affect multiple organs systems, which is a result after a loss of immunological tolerance and immune response against self-antigens [5, 6]. The diagnosis of SLE follows the 1997 updated version of the 1982 revised criteria by the American College of the rheumatology (ACR) [7, 8] where it is required that the patient have the presence of four out of eleven criteria before the diagnosis SLE is set [6, 7]. In addition to the ACR classifications the severity of SLE is assessed by the SLE disease activity index (SLEDAI), which is a scoring system that includes 24 clinical and laboratory variables that are weighted differently according to how life threatening the manifestation is [9, 10]. From these criteria it is obvious that this disease may affect some if not most of the vital organs and tissues of the body, implying it is crucial that the diagnostic tools and therapeutic agents are further developed to improve the life and health quality of the patient.

It is common knowledge that one of the major risk factor for SLE is gender, observed by that at least 9 out of 10 patients are women [6, 11, 12]. The incidence of SLE is varying worldwide depending on the geographic and ethnicity. Studies from United Kingdom (UK) and North America observed that Afro-Americans and Asians had a higher risk to develop SLE than other ethnical groups [13]. In Scandinavia the total annual incidence rate (AIR) is lower, between 2.35-3.5/100 000 [5, 10, 13, 14], compared to countries with a much wider ethnicity spectrum such at United Kingdom (total ACI; 4.7-4.9/100 000)[15, 16] and USA (Total ACI; 5.5-7.22/100 000) [17, 18]. In the study by Lerang and colleagues from 2012 the

Resolu'on

•  Ac%va%on of regula%on mechanisms

• Cell extrinsic; T_Regs

• Cell intrinsic (inhibitory pathways);

CTLA-4 and PD-1

Ini'a'on

• Gene%c factors (polymorphism)

• Environmental factors (infec%on, microbiome, UV-irradia%on)

Propaga'on

• Innﬂama%on and %ssue damage

• Cytokine produc%on

• Epitope spreading

• ShiP in the balance between eﬀector cells and regulatorical cells

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prevalence of SLE in Norway were calculated to be in total 51.8/100 000, where the prevalence for women were 91.0 and for men 10.7 [13]. At approximately the same time Lerang published her data, Hermansen and colleagues calculated the prevalence of SLE in Denmark. Interesting the prevalence from Denmark were lower than the prevalence in Norway with a total prevalence of 45.2/100 000, 79.6/100 000 for women and 10.1/100 000 for men [5].

1.2.1 Pathology SLE

The one and exact factor for the disease development in SLE is sadly still unknown, making it difficult to predict, diagnose and treat. In Figure 1.3 some of the more common factors that might stimulate disease development are illustrated, but it is wise to be aware that development of the disease often are caused by a mix of several factors [19, 20]. Figure 1.3 also illustrates some of the most common immunological effects and which organ that are associated with SLE. One of the most central immunological disturbance in SLE is the production of autoantibodies, which is an important contributor in the pathogenicity and diagnostic of the disease. Antinuclear antibodies (ANAs), which are found in 90 to 95% of SLE patients [21], are antibodies that can recognize and bind to components in the cell nucleus, such as DNA, RNA, nuclear protein, and the protein-nucleic acid complexes nucleosome and spliceosome [21, 22]. In SLE there are several factors that could contribute to the development of autoantibodies, but the deficiency of removal of apoptotic cell debris might be one of the leading mechanisms [23].

Figure 1.3: Overview of some of the pathogenically hallmarks of SLE. This illustration was inspired by “Figure 1” in the review article “Mechanisms of disease - Systemic Lupus Erythematous” published in 2011 by G.C Tsokos.

Organ or tissue damaged

Skin Kidney Heart Lung Brain/CNS Joints and bone

Effectors

Immune complexes Autoantibodies (ANAs etc...) Cytokines Activated T cells

Initiation factors

Genetically Enviromental (infaction, UV,

drug-induced) Immunoregulatory Epigenically Hormonal (progresteron, estradion)

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1.2.2 Lupus nephritis

Lupus nephritis (LN) is the kidney manifestation of the SLE autoimmune disease and is one of the more severe manifestations of SLE [21]. 25% to 60% of the patients with SLE are affected by this renal manifestation and this occurs often during the first year of disease course [10]. Classification of LN follows the classification system provided by the International Society of Nephrology and the Renal Pathology Society from 2003 (ISN/RPS 2003) [24]. This criteria system is based on the glomerular changes in LN patients, from when immune complexes deposits (ICs) in the glomerular to when sever scaring occurs and the function of the glomeruli are lost and proteinuria is observed.

The pathology of LN (Figure 1.4) is characterized by deposition of immune complexes (IC) in the glomeruli, which will lead to an inflammatory cascade with activation of Fc receptors (FcR) and Toll-like receptors (TLRs) on the cells in the glomeruli and the tubulointerstitium [20, 21, 25-27]. Activation of these receptors will stimulate the production of proinflammatory cytokines such as IL-1, IL6, TNFα and monocyte chemoattractant protein 1 (MCP-1), which again will contribute to the recruitment of immune cells [26, 28]. In addition to the production of proinflammatory cytokines, the cells in the kidney will also start the production of extracellular matrix (ECM) compounds [29]. These ECM compounds promote the scaring formation in for example the glomeruli and causes glomerulosclerosis, leading to organ failure and proteinuria [29].

Deposition of ICs in the glomeruli might also stimulate mesangial cells to proliferate and expand the mesangial matrix, leading to a reduced flow of filtrating in the glomerular capillaries and thereby eventually seal the capillary lumen [30]. The fenestrated endothelial cells in the glomeruli will also be activated and start to express adhesion molecules such as VCAM-1, ICAM-1 and E-selectin, when the ICs are deposited in the glomeruli [25, 31, 32].

These adhesions molecules are important for the recruitment and infiltration of immune cells to the subendothelium and mesangium [25, 32]. The deposition of ICs in glomeruli and with the infiltration of immune cells and expanding mesangium, add a huge stress on the specialized glomerular epithelial cells; the podocytes. One of their main responses is the loss of their characteristic pattern of foot processes in a process called effacement in podocyte foot processes [33]. In a study by Wang et al. from 2014, a positive correlation between the widths of the foot processes and the level of proteinuria was observed [34]. These findings might be explained with that the foot processes are important for the filtration processes in the glomeruli, and when they are damaged the filtration will be affected and interrupted in a way leading to proteinuria [33].

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Infiltrating leukocytes are also associated with formation of tertiary lymphoid structures (TLS) with active germinal centres (GCs), which will promote B cell differentiation into antibody secreting plasma cells and stimulate a local production of autoantibodies [26].

Chang and colleagues published in 2011 the first article where they describe how infiltrating immune cells are capable organize into B-T cell aggregates and GCs in lupus patients with nephritis [35]. From this study, they found that there was a correlation between the B-T cell aggregate and GCs formation and the IC deposition in the tubular basement membrane [35].

Figure 1.4: Some of the possible outcome when circulating immune complexes and ANAs are deposited in the kidney.

When ICs are deposited in the glomeruli or in the tubulointerstitium they will activate the cells in the tissue to produce proinflammatory cytokines and chemokines, which will recruit immune cells to the site of inflammation. The kidney cells will also have an increased production of ECM components leading to fibrosis. TLSs have been observed in kidneys of LN patients and in murine models of LN. The end outcome of the damaged kidney is eventually kidney failure with proteinuria.

Based on ref [20, 26, 29, 34, 35]

Circulating IC and ANAs (anti-dsDNA AB)

Mesangial cell Endothelium

Podocyte

Podocyte GBM

Mesangium

Glomerului

Tfh cell

Plasma B cell

GC reaction

B cell BCR

T cell

HEV Effector T cell

TLS formati on

Proinflamatory cytokines/chemokines Production of ECM proteins

Effacement foot process

Pr oteinuria

Expansion of mesangial matrix

Proinflamatory cytokines/chemokines

Infiltrating immune cells

PTEC

PTEC PTEC

PTEC

Tubulointerstitium

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1.3 Immune cells

The soldiers of the immune system are the immune cells, which are composing both the innate and adaptive immune system (Figure 1.5). The innate immune system could be viewed as the first line defence and the cells in this part of the immune system are the first one to react to a pathogen exposure [36]. The phagocytic cells, such as neutrophils and macrophages; cytotoxic natural killer cells and granulocytes will carry out the effector functions of the innate immune responses [36, 37]. The adaptive immune responses will immediate an antigen-specific defence with the development of the (long lived) antigen- specific lymphocytes; B and T cells [36, 37]. Antigen-presenting cells, which include cells from the innate immune system such as macrophages and DCs, are important in the activation and priming of the antigen-specificity of the adaptive immune system. In this study the T cells from the adaptive immune system are in focus and will therefore be discussed further.

Figure 1.5: Immune cells of the innate and adaptive immune system. The innate immune system consist of the dendritic cells, the granulocytes (basophils, eosinophils, neutrophils), macrophages, NK cells and mast cells, while the adaptive immune system consists of the antibody producing B cell, the CD4+ and CD8+ T eclls. γδ T cells and NK-T cells cytotoxic lymphocytes that straddle the interface of innate and adaptive immunity. B; B cells, BG; basophil granulocyte, DC; Dendritic cells, EG; eosinophil granulocyte, MC; Mast cell, Mφ; Macrophage, NK; natural killer cells, NKT; natural killer T cells, T; T cells, CD4+Th; CD4+ T helper cells, CD8+ CTL; CD8+ cytotoxic T cells, γδ-T; gamma delta T cells. Figure modified from Figure 1 by Dranoff [37].

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1.3.1 T cells and their main linages

T cells are lymphocytes that play an important role in cell-mediated immunity, and are characterized with their expression of their T cell receptor (TCR). These cells develop in the bone marrow and are primary matured in the thymus as discussed in the Section 1.4.1. The T cells are broadly divided into the two main linages, αβ and γδ, based on how their TCR are composed of [38]. The first combination of the TCR consist have α and β chains, and most of the T cells belong to this class. TCR of both the sublinage of CD4+ T helper cells (Th cells) and the cytotoxic CD8+ T cells (CTL) consist of α and β chains. These cells will recognize antigens presented on the MHC molecule to either assist other cells in immunological effector functions (Th Cells), or to kill infected cells and cancer cells (CTL).

In the αβ linage of T cells consists also of the Natural killer T (NKT) cells, which have both phenotypic and functional characteristics found in both conventional NK cells and αβ-T cells [39]. The NKT cells have the ability to recognize lipid antigens presented by the CD1d- molecule cell types present in the intestine and liver, and could thereby contribute to the immune responses in the digestive system for both promoting health and disease [39].

The second main linage of T cells, the γδ T cells, show several innate cell-like features that permit early activation and recognition of conserved non-peptide ligands presented by stressed cells [40]. Interesting these γδ T cells are mainly located in mucosal tissues and on epithelial surfaces, such as the gut mucosa, skin, lungs and uterus, where they migrate early in their development [40]. The main functions of the γδ T cells varies from a protective immunity against extracellular and intracellular pathogens, tumour surveillance, modulation of both innate and adaptive immune reactions to tissue healing and regulation of the function of a physiological organ [40].

1.3.2 The family of CD4+ Th cells

The CD4+Th cells are further divided in to subsets based on the differentiation of naïve CD4+ T cells, which depends on the antigen, the strength of the TCR signal and the cytokines in the environment [41] (Figure 1.6). In 1986 Mosman and Coffman distinguished Th1 and Th2 subsets form each other based on the cytokine profile of these two subsets [42].

The main functions of Th1 cells are involved in cell mediated inflammation, defence against intracellular pathogens and in delayed-type hypersensitivity reactions [43]. The Th1 cells are known for their production of the characteristic cytokines: IL-2 and IFNγ, but they can also produce other cytokines such as TNFα and LTα [43]. The T cells of the Th2 subset are

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involved in humoral-mediated immunity and their main function are to defend the host against extracellular pathogens, but unfortunately the Th2 subset are also associated with allergy, eczema and asthma [43, 44]. The characteristic cytokine profile of the Th2 subset consists of IL-4, IL-5 and IL-13, as well as IL-9 ad IL-10. IL-4 is a cytokine with several functions for other lymphocytes and for cells from the innate immune system. The IL-4 cytokine promote activation in macrophages and monocytes, stimulate development and maturation of dendritic cells, and for plasma cell differentiation and antibody isotype switching to IgG1 and IgE [43].

After the discovery of Th1 and Th2 subsets of CD4+T cells, several subsets of CD4+

T cells have been classified such as Th9, Th17 and Th22 cells, T regulatory cells (Treg) and follicular helper T cells (Tfh) [43]. The Th17 subset is characterized by its expression of IL- 17, primarily IL-17A and IL-17F, in addition to their expression of TNFα, IL-6, IL-22, IL-21 and IL-26 [43, 45]. The main function of the Th17 subset involves the host defence against extracellular bacteria, fungi and viruses [43, 45], where these cells stimulate production of antimicrobial peptides, increase the barrier function of epithelial cells and lead to recruitment of neutrophils and monocytes to the site of inflammation [46]. The Th9 subset of the CD4+ T cell repertoire are one of the main producers of IL-9, which will stimulate inflammation by promoting the growth of leukocytes such as mast cells and the secretion of chemokines that will stimulate the recruitment of more immune cells to the site [47]. In addition to their production of IL-9, the Th9 cells can also produce IL-10, which is an anti-inflammatory cytokine and indicating that Th9 cells might perform immune regulatory mechanisms [43].

The newest member to the Th subset is the Th22 cell, which has several similarities to the Th17 cell with the production of IL-22 [43]. The IL-22 is a member of the IL-10 family, indicating that the role of Th22 cells in host defence acts on non-immune cells and promote enhancement of innate immunity and tissue regeneration [43, 48].

The linage of Treg cells are a subset of specialized T cells that execute suppressive functions of other effector T cells and could be seen as a “police” or a “break” of the immune system. Their main task is to control and supress overactive immune cells [43]. Some examples where they have an important role are: their suppression of allergy; in the establishing of tolerance to organ grafts and to prevent graft-versus-host disease; and to promote feto-maternal tolerance in pregnant women [49]. αβTCR linage of Treg cells are characterized by their transcription factor FoxP3 and can be developed in the thymus (nTregs), as a result of central tolerance, or they could be induced via post-thymic maturation

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(iTreg) that often are characterized as CD4+CD25+FoxP3+ [43]. The Tregs uses multiple methods to perform their effector functions: one through modulation of APCs and thereby indirectly supress T effector function, or by directly suppress T and B cells [50]. Treg cells can secrete of anti-inflammatory cytokines (IL-10 and TGFβ), which will inhibit the proinflammatory effector functions of lymphocytes and APCs [50]. Treg cells are also capable with expression of inhibitory receptors (CTLA-4) [43], which can down regulate the expression of MHC-II molecule and the costimulatory molecules CD80/CD86 and CD28 on the APC [43, 49]. The Treg can induce apoptosis in T cells or APCs through cell-to-cell contact by a granzyme or perforin mechanism, or via the stimulation of tryptophan catabolism in APCs through indoleamine 2,3-dioxygenas (IDO) that produce the T cell toxic compound kynurenines [49].

The follicular helper T cells (Tfh) are a specialized subset of T cells whose main task is to provide B cell help in the GCs together with follicular dendritic cells (FDCs) [51].

Through their expression of surface molecules and chemokines such as; CD40L, IL-21, IL-4, PD-1, and BAFF, they will regulate the B cell survival and proliferation, participate in the initiation of somatic hypermutation and differentiation of B cells into plasma B cells and memory B cells [51]. The Tfh can also induce apoptosis via Fas-FasL interactions in B cells that fail to present cognate antigen [51].

Figure 1.6: The CD4+ Th subsets; their inducing cytokines, their expression of transcription factor, and their main production of chemokines. The chemokines and transcription factors included are only some of the factors that will determine the fate of the naïve CD4+ T cell. Information from Table 1 by Tangye et.al (2013) [48]

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1.3.3 CD4+ T helper cells and their role in lupus

It is observed that T cells from patients have abnormal phenotypes and functions [52], which can lead to exaggeration of TCR response to a stimuli and the T cells get activated [19, 53]. The CD4+ T helper cells are important in the production of autoantibodies and tissue inflammation, and they have a strong connection to the pathogenicity of SLE and LN [54]

(Figure 1.7). All the cytokines produced by Th1 and Th2 are important for the pathogenicity of SLE and lupus prone mice that are IFNγ^-/-and IL-4 ^-/-have reduced lymphadenopathy and end-organ disease compared to the cytokine sufficient control group [55]. In murine models of SLE, deficiency in the IFNγ and IL-4 have shown to be important for the pathology of the disease [43, 56].

Today it is clear that other subsets of T cells, both within the Th family and in other subset of T cells, are of high importance in the contribution to the immune disturbance in SLE [6, 57]. An increase in the IL-17-levels in the blood and tissues (kidney, skin) from lupus patients have been observed [54], indicating that the Th17 cells are involved in the pathogenicity of lupus. From murine models IL-17 have shown to promote spontaneous formation of ectopic GCs, stimulate loss of B-cell tolerance and maturation of B cells into plasma cells, in addition to induce autoantibody production in these B cells [43, 54, 56]. IL - 17 have also been associated with infiltration of NK cells and neutrophils, and an increased IFNγ production by NK cells, CTLs and Th1 cells has been detected in patients with nephritis [56]. Circulating follicular helper-like CD4+ T (cTfh-like) cells have been observed to be associated whit the disease activity in SLE patients were, which can indicate that the regulation of the maturation of naïve B cell might be disturbed and promote the development of ectopic GCs [58, 59]. The development of the different Th effector-subsets and the Tfh are under control of Treg cells [49]. If the balance between the effector and regulatory cells is disrupted, the chances of developing autoimmune diseases are increased [49]. Impaired functions and reduced numbers of Treg cells in patients with lupus have been reported, and are linked to the disease progression in SLE (reviewed in [60]). In 2005, Hatashi and colleagues found depletion of Treg cells in murine models resulted in increased titers of ANAs and an early development of glomerulonephritis compared to the control [61]. These results support the importance of Treg in the control of effector functions of the immune system.

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Figure 1.7: Dysregulation of T cell function and subset population in SLE pathogenesis. Reduced effector functions of the Treg and the CD8+ CTL stimulate an increase in pro-inflammatory Th and Tfh subsets, which infiltrate tissues, enhance the inflammation processes, and stimulate autoantibody production. DN-T cells are also observed to contribute to the disease pathology through their production of IL-17. Green arrows indicating upregulated pathway and red arrows indicating down regulated pathway. Modified from figure 1 from Suárez-Fueyo et.al (2016) [54]

1.4 Lymphoid organs

Even though good defences against infections are scattered in tissues throughout the body, the optimized structures to create pathogen specific lymphocytes include the lymphoid organs. These organs are important for the production and activation of “combat approved”

lymphocytes, which during normal conditions are not self-reactive. The lymphoid organs can be divided into three main categories; primary, secondary and tertiary lymphoid organs or tissues, and their main function and development will be discussed in this section.

1.4.1 Primary lymphoid organs

Primary lymphoid organs are defined as organs, or compartments within organs, where hematopoietic progenitor cells differentiate into an abundance of immune cells capable of performing effector functions [62]. There are three main types of primary lymphoid organs:

the bone marrow, the thymus and the fetal liver, which will follow a programmed development during embryogenesis [62]. In addition to produce immune cells with effector function, the primary lymphoid organs also hold the site of central tolerance mechanisms (Figure 1.1).

In the adults the bone marrow harbour the source of self-renewing stem cells such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells, in addition to various progenitor cells that have started their pathway in differentiation

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and specialization into specific cell types [62, 63]. One of the main functions of the bone marrow is to produce erythrocytes, platelets and leukocytes such as neutrophils, monocytes and mast cells, just to mention some of the common leukocytes. The bone marrow is also the site of B cell maturation, where IgM producing B cells are developed before they are distributed into the blood. The central tolerance of B cell maturation occurs in the bone marrow, where a combination of positive and negative signals from the BCR and co-receptors together with signals from survival factors (i.e. BAFF) will determine the fate of the B cell [2, 64]. The three outcomes of these signals are the survival and activation of the developing B cell, the clonal deletion by apoptosis or the B cell will reach an inactivated anergic phase.

Self-reactive B cells can undergo receptor editing, where an additional light chain VJ recombination and new Ig light chain production occurs, in the hope of changing the BCR specificity not self-reactive. If the outcome of the receptor editing still creates a self-reactive B cell, the B cell dies via apoptosis [64].

The thymus is the main location where lymphocyte progenitors cell undergo a multistep maturation, differentiation, expansion and selection program to become either a naïve CD4+ or CD8+ T cell [62], which can be activated in secondary lymphoid organs and mediate cellular immunity. In this maturation program of T cells, there are some essential checkpoints that the developing T cell has to pass before it is released into the circulation as mature naïve CD4+ or CD8+ T cell. T progenitor cells (thymocytes) entry the thymus in the corticomedullary junction and start their journey through out the thymus [65]. At this stage the thymocyte lack the expression of the TCR, CD4 and CD8, and are termed double-negative (DN) thymocytes. In the cortex of the thymus these DN thymocytes goes through four stages of differentiation and simultaneously starts to express their pre-TCR molecule [66]. When the thymocyte manages to successfully express the pre-TCR, the thymocyte will proliferate and become double positive (DP) thymocytes with their expression of CD4 and CD8 [62]. Then the TCR on the DP thymocytes will interact with peptide-MHC complexes that are expressed by cortex stromal cells, such as cortical thymic epithelial cells (cTEC) and DCs in the cortex.

Low-avidity interactions will induce the DP thymocytes to receive signals for survival and differentiation into single positive (SP) thymocytes [65]. The next event in the developing thymocyte is the central tolerance, which occurs in the medulla of the thymus. Here the autoimmune regulator (AIRE) expressing medullary thymic epithelial cells (mTEC) will interact with the SP thymocytes through peptide-MHC complexes, and recognize thymocytes which binds to strongly to the complex [65]. These self-reactive thymocytes will either fate death by apoptosis or be stimulated to become FoxP3+ nTreg cells [65]. Sphignosine-1-

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phosphate receptor 1(S1P1) expressing mature T cell, which have overcome all the developing checkpoints in the thymus, will be attracted to the circulation where they can be activated and become effector T cells [65].

1.4.2 Secondary lymphoid tissues

Lymph nodes, spleen, and mucosa associated lymphoid tissues (MALTs), such as payer’s patches (PPs) and tonsils, are common known as secondary lymphoid organs (SLOs) located statically within the lymph and blood [62, 67, 68]. The organization of the immune cells share several similarities

The lymph node is a highly organized organ that is surrounded by a capsule, and the three main compartments are the cortex, the paracortex and the medulla [62, 69]. The cortex contains B cells, macrophages and follicular dendritic cells (FDCs) arranged into primary B cell follicles, where the chemokine CXCL13 produced by the FDCs plays the dominant role in the position of the B cells via the interaction with CXCR5 [62]. In the primary follicles B cells immunity are mediated by FDCs [70]. These FDCs can present antigens in form of immune complexes that are bound via Fc and complement receptor, and thereby stimulate B cells through the BCR receptor and promoting germinal centre formation [70]. The T cell zone located in the paracortex are mainly composted of T cells, DCs and fibroblastic reticular cells (FRCs), where the chemokines CCL19, CCL21 and CXCL12 are important for the organization and recruitments of T cells and DCs [69]. It is in the T cell zone naïve T cells are activated by antigen-presenting cells APCs, which in most cases are mature DCs. In between the cortex and paracortex are the location of the secondary B cell follicles and germinal centres (GCs), where Tfh cells are involved in somatic hypermutation and isotope switch of the B cells Ig-molecule [71]. The most inner layer of the lymph node, the medulla, consists of the medullary cords that are separated by the lymph filled spaced of the medullary sinuses [69]. In the lymphatic sinuses, the filtration of lymph from afferent lymph vessels occurs, before the compounds from the lymph flows to the B cell area in the cortex or via the subscapular sinuses and out of the lymph node via the efferent lymphatic vessel [71]. The afferent and the efferent lymphatic vessels lined with lymphatic endothelial cells, together with the high endothelial venules (HEVs), complete the vasculation of the lymph node [71].

B and T cells will, together with blood antigens, enter the lymph node via HEVs in a process known as the leukocyte adhesion cascade [62]. The HEVs in the lymph node express a special selectin ligand called pheripheral LN adressin (PNAd) on their luminal surface,

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which will interact with the L-selectin (CD62L) on B and T cells and initiate the rolling cascade. The chemokines located at the HEVs; CCL19, CCL21, CXCL12 and CXCL13, are important for guiding and select the B and T cell via their chemokine receptors (CCR7, CXCR4 and CXCR5) into the lymph node. These chemokines will activate the chemokine- triggered adhesion to the HEVs, which involves activation of the α1β2 integrin to ICAM1/ICAM2 on the HEVs. [62]

Immunological properties

The main functions of the SLOs are to filter the blood and lymph to trap and concentrate foreign antigens in addition to attract antigen-presenting cells (APCs), which have brought in antigens from surrounding tissues, to initiate an adaptive immune response with activation of naïve lymphocytes [62, 63, 67, 72]. Second important functions of the SLOs are their capacity to execute mechanisms for peripheral tolerance. There are several ways that the tolerance mechanisms are archived, and are involved with several cell types inside the SLOs.

The essential parts in the decision of the fate of the naïve lymphocytes lies in the presence or absence of antigen, co-stimulation, cell interaction and/or chemokines/cytokine [69].

In the classical activation of naïve T cells, the naïve lymphocyte needs two types of signal to be fully activated. The first signal is depended of the TCR:MHC interaction between the T cell and the APCs, while the second signal is provided to the T cell by the APC and it is composed of costimulatory molecules such as CD80/CD86 and chemokines [73]. Usually the APC that are involved in this process are mature DCs, which have migrated from surrounding tissues after they have been activated by a “danger signal” via their pattern recognition receptors (PRRs) and captured antigens. In for example the lymph node, there are several immature DCs that have not been activated but can present antigens on their MHC-II molecules [74]. If the MHC-II molecule on this immature DC interacts with the TCR on a naïve CD4+ T cell, the T cell either dies or become anergic since the immature DCs can’t provide with the important second signal with the expression of costimulatory molecules [74, 75].

Initially stromal cells were thought to be mainly involved in supporting of the structure of SLO [69, 75]. However, later research has found that they can also serve an important immunological function in the regulation of the adaptive immune system [69, 75].

These stromal cells, especially the fibroblastic reticular cells (FRCs), can express peripheral tissue-restricted antigen (PTAs), such as proteins associated with pancreas, eye, intestine,

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thyroid, skin, CNS and liver, in a similar fashion as the mTECs does in the thymus [76]. The PTA expression has shown to induce anergy and subsequent elimination of CD8+ T cells, and might be a method to increase the odds for eliminating rare, self-reactive T cells [76].

Development of secondary lymphoid organs

The development of the SLOs is a pre-programmed process and occur either during the embryogenesis or early in the post-natal period [68]. The SLO development requires the interaction between the mesenchymal stromal cell expressing the lymphotoxin-β receptor (LTβR) and the hematopoietic lymphoid tissue inducer (LTi) cell, which express the lymphotoxin-α1β2 (LTα1β2) complex [70, 72]. LTα1β2 is a heterotrimeric complex belonging to the TNF superfamily, it is composed of the membrane-bound LTβ2 and the soluble LTα1 and by binding to its receptor LTβR will initiate a signalling cascade necessary for the developing lymphoid organ and the interaction is necessary for the maintenance of the organized structure [77].

The LTi cells are derived from the family of type 3 innate lymphoid cells (ILC3) and are characterized by their expression of being ID2+RORγt+CD4+ [78, 79]. The development of the LTi cells from the ILC3 is strictly depended on their expression of the transcription factors RORγt and the ID2 and the TNF family ligand-receptor pair RANKL- RANK/TNFRSF11A [79], where the RORγt expression are controlled by maternal retinoic acid [78]. Figure 1.8 illustrates the main events that occur during SLO development. Before the interaction between the LTo cells and the LTi cells occurs, the LTi cells need to be clustered in a LTα1β2/LTβR independent manner [72, 80].

The chemokine CXCL13 produced by the mesenchymal stromal cells are important for the initial clustering of LTi cells by binding to the CXCR5 on these cells [81], and activation of the CXCR5 results in increased levels of LTαβ on their cell surface [79]. In mice with deficient CXCL13 and CXCR5 signalling have an insufficient development of peripheral lymph nodes [81] and the white pulp in spleen [82]. The production of the CXCL13 chemokine is under the control of retinoic acid from neurons, observed by retinoic acid producing neurons are located near to CXCL13 expressing stromal cells near the branching site of blood vessels [81]. Retinoic acid will control the gene expression of CXCL13 by binding to DNA-binding RA receptors (i.e. RARβ), which then will bind to the retinoic-acid-responsive elements (RARE) in the CXCL13 gene and induce the CXCL13 production [81].

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The increased expression of LTαβ on the LTi cells promote the LTα1β2/LTβR interaction between the LTi and the mesenchymal stromal cells, and results in maturation of the mesenchymal stromal cell into lymphoid tissue organizer cell (LTo). The LTo are characterized with their expression of the adhesion molecules (VCAM-1, ICAM-1 and MADCAM-1) and their increased production of the homeostatic chemokines (CXCL13, CCL19 and CCL21) [72, 79, 80]. The LTo cells will also produce interleukin-7 (IL7), which together with the homeostatic chemokine upregulation lead to a positive feedback loop that will result in an increased recruitment of LTi cells [80]. The LTo cell will also contribute to the incorporation of lymphatic endothelial cells (LECs) by expressing VEGF-C [62]. LTo cells secreting potent vascular growth factors will stimulate LTβR expression on endothelial cells, which are important for the differentiation into HEVs [83]. In addition the LTβR signalling is necessary for maintenance and homeostasis of this HEV network in the developing SLO [83]. The LTo will later in the SLO development differentiate into the non- haematopoietic stromal cell types such as FDCs and FRCs [80]. In development of PPs in the small intestine, a second distinct population of CD4-CD45+ ILRα- CD11c+ lymphoid tissue induction (LTin) cells are involved in the stromal activation in the developing organ [80].

Figure 1.8: The first events in the developing SLO.1. Retinoic acid from nerve cell will stimulate mesenchymal stromal cell to produce CXCL13, which will interact on the CXCR5 receptor on a LTi cell (2.). The LTi cell will respond with production of LTαβ (3.), which will interact with the LTβR on the stromal cell and stimulate differentiation into LTo cell (4.). The LTo cell will produce a positive feedback and recruit more LTi cells to the site by producing CCL19 and CCL21 (5.). The LTo cell will also contribute the formation of HEVs and incorporation of lymphatic endothelial cells (LECs) to the structure (6.). In the later stages of the developing SLO, the LTo cell will further differentiate into FRCs and FDCs (7.)

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1.4.3 Tertiary lymphoid structures

Tertiary lymphoid structures (TLS) are ectopic accumulation of lymphoid, myeloid and stromal cells, which occur after birth and are often observed as a response to an environmental stimuli and/or the transition from an acute to a chronic inflammation [62, 79].

TLSs are observed in a large variety of diseases ranging from several autoimmune diseases [84], cancer [85], infections [86], to transplant graft rejections [86]. How the TLS is organized varies between the site of inflammation, organ affected and individual variance between each patient. The degree of organization ranges from oligocellular accumulation of B and T cells to more sophisticated structures similar to SLO with distinct B and T cell area, active GCs, PNAd+ or MAdCAM-1+ HEVs, FRCs and FDCs [62]. The TLS formation is a reversible process, observed with that they can decrease in size after the removal of the initiating stimulus or after therapeutic intervention, and thus making TLSs to be a quite plastic lymphoid organ [67].

The development of TLS, or the neogenesis of the TLS, shows several similarities to the SLO development regarding the chemokine/cytokine and adhesion molecule expression patterns [79]. One of the most dominant similarities is the expression of homeostatic chemokines (CXCL13, CCL19, CCL21 and CXCL12) and signalling through LTα1β2/LTβR leading to the positive feedback loop that guide the recruitment and organization of lymphocytes [87]. The more striking differences between the SLO and TLS development are that the TLS development does not require fetal derived CD4+CD3-RORγ+Id2+ LTi cells and inflammatory events are triggers for the TLS development, while the SLO development is depended of fetal derived LTi and is pre-programmed during embryogenesis [87]. The questions then arise regarding which cell type can function as an LTi cell candidate in the developing TLS. Infiltrated immune cells, such as DCs, macrophages, Th17 cells and γδ T cells could function as an LTi-like cell and express LTα1β2 [77, 86]. Recent studies have shown that the cytokine IL-17, which are produced by Th17 and γδ T cells, are important in the initiation of TLS structures [77]. The cells that can act as an LTo cell candidate in the developing TLS are the stromal cells (from a mesenchymal progenitor cell) in the inflamed tissue; the fibroblasts, myofibroblasts and the perivascular cells, which under stimulation by proinflammatory cytokines can express LTβR and produce compounds for recruitments of LTi-like cells leading to the formation of TLS [88]

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Tertiary lymphoid and diseases

What outcomes, both beneficial and detrimental, are associated with TLS formation in a host? To answer this question the case studied has to be defined, since there are both pros and cons with this ectopic lymphoid tissue development. In viral infections, such as in acute influenza virus infection, bronchial-associated lymphoid tissue (iBALT) formation in the lower respiratory tract is observed and induces a host protective role against the infection [89].

On the opposite site of the scale, the development of TLS might be the cause of the induction or exacerbating of an autoimmune reactions and thereby is associated the detrimental effects [80]. Activation of autoreactive B cells in TLS is not so strictly regulated compared to the activation of B cells in the SLOs, increasing the risk of differentiation and expansion of these autoreactive B cells and promote a local production of autoantibodies in the inflamed tissue [90]. TLSs have been associated with several autoimmune diseases and where these structures develop are related to where the immune system usually attack in the disease [89]. For example TLSs have been observed in the joints and lung of patients suffering of rheumatoid arthritis [91], in the salivary glands in Sjögren’s syndrome [92], in the pancreas in diabetes [93], and in the kidney of SLE patients [35]. TLS formation in patients might also affect how they are responding to the therapy, making it challenging to treat this patient group [90].

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1.5 Stem cells

Stem cells are characterized with their differentiation potential to become multiple mature cell types and their ability to self-renewal, which are important to replenish the stem cell pool [94, 95]. The ability to differentiate into various cell types is described with the potency of the cell. The more cell types a stem cell can be the ancestor of, the higher is its potency, which can range from a totipotent (ie. Zygote) to unipotent (ie. spermatogonial stem cells) [94]. The stem cells can broadly be divided into two main categories; the embryo stem cells and the adult somatic stem cells, also known as the nonembryonic stem cells [96]. The embryo stem cells are isolated from the inner mass of a blastocyst and are derived from the totipotent zygote [94, 96]. The embryotic stem cells, categorized as pluripotent stem cells, have the ability to become the ancestor of all the cells in a developing fetus and some of the extra embryonic cells such as cells in the placenta [94]. The adult somatic stem cells can be found in adults and children, but also in the infants, placenta and the umbilical cord blood [96]. These adult stem cells are known to be multipotent, meaning that they have the capacity to generate the mature cell type of their tissue origin, but will not differentiate into unrelated linages under normal physiological circumstances [94]. The bone marrow harbours two types of adult somatic stem cells; the hematopoietic stem cell (HSCs) and the mesenchymal stem cell (MSCs) [96]. In this study MSCs are in focus, and their functions and role in medicine are further discussed.

1.5.1 Mesenchymal stem cells

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are nonhematopoietic stromal cells that have the potential to differentiate into tissues of mesenchymal origin such as bone, cartilage, adipose, connective tissue, smooth muscle and hematopoietic supporting stroma [97, 98]. Isolation of MSCs have been successfully performed from various tissues such as bone marrow, adipose tissue, nervous tissue, placenta, menstrual blood and dental pulps [99, 100]. A challenge in the field of the study of MSCs have been the lack of one uniform specific marker, but the MSCs cells do express patterns of surface markers [99, 100]. Table 1.1, present some of the common surface molecules that MSCs are expressing [100], but the MSCs should also lack the expression of CD45, CD34, CD14, CD11b, CD79a, CD19 and HLA-DR (MHC-II) surface molecule [101].

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Table 1.1: Some of the surface molecules expressed by MSCs, table modified from Xie 2015 [100]

Surface molecules expressed by MSCs Other cell types expressing the surface molecule

Stro-1 Endothelial cells

Sca-1 HSCs, cancer stem cells

CD13 Cancer stem cells, myeloid cells

CD29 Neural stem cells, cancer stem cells

CD44 T cells, cancer stem cells

CD73 Endothelial cells, lymphocytes

CD90 (Thy-1) T cells

CD105 (Endoglin) HSCs, endothelial cells, macrophages

CD106 (VCAM-1) Endothelial cells

CD146 (MCAM) T cells, pericytes, endothelial cells

CD166 (ALCAM) Epithelial cells

CD271 (LNGFR) Neural stem cells, cancer stem cells

Nestin Endothelial progenitor cells, endothelial cells, fibroblasts

PDGFR-α (CD140a) Fibroblasts, smooth muscle cells

Leptin-R Adipocytes

In addition to their ability to differentiate into several cell types, the MSCs exhibit the immuno regulatory capacity of immune cells [102]. This characteristics have made these cells interesting in the development for treatment of immune-mediated disorders [102]. The immune phenotype of the MSCs is considered as non-immunogenic, characterised as MHC I+, MHC II-, CD40-, CD80- and CD86-, and transplantation into an allogeneic host may not lead to an allogeneic response [97, 102]. MSCs activated in a milieu with high levels of IFNγ, TNF-α, IL-1α and IL-1β have shown to stimulate the immunosuppressive mechanisms of MSCs, which can supress the effector functions of macrophages, neutrophils, NK cells, DCs, T cells and B cells (Figure 1.9) [103, 104]. Some of the secreted compounds that MSCs use in their function in immunosupressive mechanisms are IL-10, TGF-β, nitric oxide (NO), catabolites of IDO activity (i.e. kynurenine), Tumor necrosis factor-inducible gene 6 protein (TSG6), and prostaglandin E2 (PGE2) [103, 104]. These compounds will stimulate the differentiation of M2 macrophages from monocytes, which are important for tissue repair and have anti-inflammatory properties with its enhanced production of IL-10 and TGF-β [104]. In addition the MSCs will also stimulate the recruitment of monocytes and macrophages to the site, through their production of CCL2, CCL3 and CCL12, thus enhance the differentiation of more M2 macrophages [104]. With their production of catabolites of the IDO activity and PGE2, the MSCs will both supresses T cell proliferation (arresting in the G0/G1 phase of cell cycle) and favour the iTreg differentiation [104, 105]. MSCs can induce a cytokine profile shift in Th1-Th2 balance towards the Th2 subset of CD4+ T cells [106].

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Environments with weak inflammation have paradoxically shown to stimulate MSCs and enhance immune responses by stimulating T effector cell function and differentiation of the proinflammatory M1 macrophage [103, 104]. In an early phase of inflammation or during chronically inflammation, the proinflammatory activities of MSCs can be beneficial in creating a proper immune response (Figure 1.9) [104]. When MSCs are exposed to low levels of the proinflammatory cytokines, such as TNFα and IFNγ, they can produce the chemokines CXCL9, CXCL10 and CXCL11 [104]. This response is observed to occur in mice, when NO production is insufficient [107]. One theory of how MSC can be polarized toward proinflammatory or anti-inflammatory phenotypes is through their activation of TLRs, this process is common known as “licensing” [108]. TLR-4 and TLR-2 are toll like receptors that will recognize components of the bacteria wall: the lipopolysaccharide (LPS)-layer from gram-negative bacteria for TLR-4 activation and lipoproteins from gram-positive bacteria for TLR-2 activation [104, 108, 109]. Priming of TLR-4 or TLR-2 priming has shown to promote the proinflammatory properties of MSCs, which will start to produce of proinflammatory cytokines such as IL-6 and IL-8 [104, 108, 109]. Contrary, activation of TLR-3, by virus dsRNA, have shown to promote the anti-inflammatory properties of the MSCs [108].

Figure 1.9: Role of MSCs in tissue repair and chronic inflammation. Recent studies on MSC-mediated immunoregulation suggest that MSCs are recruited to sites of tissue damage and activated by local inflammatory cytokines produced by activated immune cells. Depending on the types of immune responses (acute vs. chronic inflammation), MSCs may either attenuate the inflammatory response and lead to repair of the damaged tissue, or maintain a persistent chronic inflammatory response, leading to fibrosis and deformation of tissue architecture. Reused with permission from Shi et al. [110]

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1.5.2 Mesenchymal stem cells in treatment of SLE

With their immunosuppressive properties the MSCs have been studied as a candidate in therapy for autoimmune diseases such as SLE [111]. In a small pilot study from 2010 (n=15), administration of MSCs had a positive effect in improvement of the disease [112].

After one year only two of the patients had relapse of proteinuria, while the rest had a significantly decrease in disease activity and an improvement of the levels of Treg cells [112].

In an article published in 2013 by Wang et.al, promising results were reported in patients with severe SLE (n=87) treated with MSCs derived from the bone marrow and the umbilical cord [113]. After 4 years of studying the clinical effects of transplantation, 50% of the treated patents had entered clinical remission, although 23% had suffered from disease relapse [113].

In a smaller study from the same research group published in 2014, 40 patients with active SLE got intravenously transplantation with umbilical cord MSCs on day 0 and 7. After one year 32,5% of the patients reported major clinical response to the treatment and 27,5%

reported a partial clinical response, while 17,5% patients suffered with disease relapse [114].

In this study an improvement of the CD4+FoxP3+ Treg cell levels were observed 3 month after transplantation, in addition the urinary protein levels were decreased [114]. In a small pilot study from 2010 (n=15), administration of MSCs had a positive effect in improvement of the disease [112]. After one year only two of the patients had relapse of proteinuria, while the rest had a significantly decrease in disease activity and an improvement of the levels of Treg cells [112].

In murine models of SLE, MSCs treatments have also reported in promising effects with suppression of immune reactions and disease recovery [115-117]. In the study published by Ma et.al [116], reported that MRL/lpr mice treated with murine derived MSCs had an increased probability of surviving compared with the untreated control group. They could also reported that the treated mice had smaller spleens than control animals, with fewer activated Th1, Th2, B cells and plasma cells, in addition to a decreased production of anti-dsDNA autoantibodies and proteinuria [116]. The treatment of MSCs in the murine model NZBW-F1, on the other hand, was shown to increase the severity of disease and stimulate anti-dsDNA autoantibody production [118]. After treatment the mice had increased levels of plasma cells in the bone marrow, increased levels of deposited glomerular immune complexes and sever proteinuria compared to the untreated mice [118].

The Immunostimulatory Potential of Mesenchymal Stem Cells for formation of Tertiary Lymphoid Structures in Lupus Nephritis